The design of peptides that would readily be transported transcelluarly across absorptive epithelia has eluded peptide chemists for many years. This research explores an alternative approach whose long-term GOAL is to establish guidelines for the design of peptides that would instinctively open tight junctions by modulating ion transport processes, thereby facilitating their passage in the heretofore neglected paracellular pathway. 4-Phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide, m.w. 777) will be used as a model peptide. The proposed research is built upon our encouraging preliminary finding that this hydrophilic, proteolytically labile peptide unexpectedly penetrated all intestinal segments of the rabbit exceedingly well when compared with more lipophilic nonpeptide drugs. We hypothesize that this peptide facilitates its own penetration by enhancing tight Junctional permeability. This peptide warrants further investigation because it seems to contain the crucial information to guide its preferential paracellular transport in the intestinal epithelia by a novel mechanism, namely, modulation of amiloride-sensitive Na+ channels. There are 4 SPECIFIC AIMS in this 3- year proposal: (l) To demonstrate that Pz-peptide indeed penetrates the ileum and descending colon of the rabbit paracellularly, two regions with distinct biochemical and electrophysiological properties. The experimental parameters monitored will be changes in tight junctional resistance and paracellular marker transport; (2) To determine the structural features in Pz-peptide important to its paracellular penetration, using the Pz- and non-Pz-forms of 8 Pz-peptide analogs that vary in number of amino acid residues and C-terminal amino acid; (3) To determine whether Pz-peptide acts on the amiloride-sensitive Na+ channel to facilitate paracellular transport in the descending colon; and (4) To determine whether Pz-peptide elicits intracellular changes in Ca2+ and associated second messengers in the descending colon as part of the tight junctional opening cascade. The principal METHODOLOGY includes (1) use of voltage-clamp/Ussing chamber assembly to evaluate electrophysiological and permeability changes; (2) confocal laser scanning microscopy to visualize fluorescent marker transport in the intercellular space; and (3) measurement of intestinal Na+ uptake and water flux associated with paracellular Pz-peptide transport.